Digital imaging as a detector for generic analytical measurements

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Digital imaging as a detector for genericanalytical measurementsLiam Byrne, John Barker, Gaeëlle Pennarun-Thomas, Dermot Diamond*National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland

Steve EdwardsKodak Ltd., Harrow, UK

Digital imaging was investigated as a potentialmeans of processing high density dot arrays foranalytical purposes. Using a high resolution digitalcamera, a colour image of the dot array was takenand subsequently processed using in-house devel-oped National Instruments LabVIEW0 software toreturn numerical values for the red, green and bluecomponents of the colour of each dot. z2000Elsevier Science B.V. All rights reserved.

Keywords: CCD camera; Digital imaging; Dot arrays;LabVIEW; Colour analysis

1. Introduction

Until the recent advent of instrumental methodsof analysis, the vast bulk of assays were performedon the basis of reagent-based colorimetric tests,either in the form of qualitative spot tests or quanti-tative titrations. While instrumental methods havealmost completely replaced titrations, spot testsmaintain their popularity as screening methodsdue to their ease of use and low operating cost,combined with high throughput capabilities. How-ever, such tests have a number of well-knowndrawbacks such as:

õ Subjective nature of colour estimationsõ Influence of background lightingõ Impact of colour blindness on the type of colours

that may be usedõ Little scope for quantitative measurementsõ No compatibility with electronic report genera-

tion

Recently, there has been a rapid improvement inthe technology of digital photography, both interms of hardware and software performance. Thestrong indications are that this rapid developmentwill continue over the coming years, fuelled by theneed for cheap, high performance image acquisi-tion for consumer products and the Web [ 1^3 ]. Thecombination of digital photography and colouri-metric tests potentially offers a route to highthroughput qualitative and quantitative analyticalmeasurements.

1.1. Digital cameras

Digital cameras are based upon the use ofcharge-coupled devices, CCDs [ 4 ]. The pace ofthe development of digital imaging technologiesis such that digital cameras are now available onthe high-street for prices sub-US$500. For example,the image of the standard ruler taken in Fig. 1 wastaken with a high-street camera with a CCD array ofapproximately 1.3 M pixels. The image has a reso-lution of 286U193 pixels and a 1 cm horizontalsample taken of the image contains 227 pixels, asshown in Fig. 1. The resolution of the image can befurther increased by incorporating an optical lenssystem ( macrolens or microscope objective) tomagnify further the object. Each of these pixelsacts as an independent 24-bit colour sensor thatcan convert the image colour into crude spectro-scopic components.

Hence, a digital camera can act as an analyticaldetector, and generate a vast amount of informationper image.

The setup in use here is shown in Fig. 2. It is amodi¢cation of the setup used by Hislip et al. [ 5 ],who showed the application of a CCD-based digitalcamera to imaging 96-well microplates. This simplesetup allows acquisition of a desired image simplyand easily.

0165-9936/00/$ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 - 9 9 3 6 ( 0 0 ) 0 0 0 1 9 - 4

*Corresponding author. Tel.: +353 (1) 7045404;Fax: +353 (1) 7048021. E-mail: [email protected]

trends in analytical chemistry, vol. 19, no. 8, 2000 517

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1.2. Digital colour

The analytical data that a digital camera returnsare a standard trichromatic response, with 8-bit red,green and blue channels, respectively. Hence, avalue is returned to the user ranging from 0 to 255for each channel. The spectral power distribution ofthe light source and the spectral re£ectance curveof the object being analysed both play an importantrole in the value of the colour obtained througheach channel [ 6 ]. Digital colour varies somewhatto spectroscopic colour and an understanding ofthis is required in order to appreciate the coloursignal processing techniques performed by theCCD array.

The colours returned by each of the three chan-nels are referred to as subtractive colour [ 7 ]. Theyare so-called because they selectively absorb (sub-tract ) certain wavelengths of light, hence affectingthe colour seen. Thus, the spectral characteristicsare further in£uenced by the spectral power distri-bution of the light source used. It should be notedthat the eye sees the converse of the colour compo-nent that is primarily absorbed, e.g. if an objectappears red, it is because this colour is beingre£ected or transmitted by the object and the bluepart of the spectrum is being absorbed. This is alsofurther explained by the difference between lightand colour dyes: adding red, green and blue light inequal proportions produces white, while addingcyan, yellow and magenta dyes (also called colour-ants ) in equal proportions produces black.

1.3. Volume dispensing

Recent improvements in liquid handling [ 8 ] andsurface immobilisation of reagents [ 9,10 ] offer theopportunity of immobilisation of large numbers ofanalytical dots of a substrate surface. Using a micro-plotting device with X^Y positioning developed byKodak, a series of ink dots were deposited ontophotographic paper in grids composed of 7U7microdots. This device consists of reservoir cham-bers which connect to multiple dispensing tips. Theindividual microdots are approximately 500 Wm indiameter with 500 Wm spacing between the dots.The density of dots is one dot per mm2 (see Fig.3). From the earlier calculation (Fig. 1) whichshowed that there are 227 pixels per cm, it wascalculated that there are approximately 102 pixelsper dot. The software used targets an area of 100pixels in size; given the 1.3 M pixels per image, it isclear that a vast amount of analytical data can beobtained per image.

Hence, the need for an analytical detection sys-tem that can quickly and readily analyse large vol-umes of data and extract the relevant information.This is as much a feature of the software in use as thehardware used for image acquisition.

1.4. Software

The use of digital images opens the possibility ofincreased usage of processing by computer soft-ware without the need for cumbersome and time-

Fig. 1. Image of ruler acquired with digital camcorder. Num-ber of pixels per cm is shown.

Fig. 2. Schematic of digital image acquisition system.

518 trends in analytical chemistry, vol. 19, no. 8, 2000

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consuming scanning of images created by conven-tional photographic techniques.

Software was developed in-house using NationalInstruments LabVIEW0 and National InstrumentsIMAQ Vision0 software [ 11 ]. The objective of thesoftware was to read an image from a selected ¢le,locate all objects which match a predeterminedtemplate which was `learned' for comparison, ana-lyse the RGB values of each of the objects, and thenoutput the data obtained to a ¢le for analysis. A10U10 pixel region around the centre of each dotwas analysed for RGB components and standarddeviations. The program returned the values forthe red, green and blue channels as well as thestandard deviations of each channel. MicrosoftExcel0 was then used to process the data byusing a pre-recorded macro. A £owchart showingthe steps performed by the software is shown inFig. 4.

2. Results and discussion

2.1. Software validation ( Fig. 5 )

A 7U7 aqua ( blue) dot array (Fig. 5, 1a) and a7U7 aqua array including blocks of red and twodarker blues (Fig. 5, 1b) were graphically gener-ated. Each of these dots has a diameter of 33 pixels;hence, there are approximately 855 pixels per dot.These dot arrays were imaged and analysed by thein-house developed software. Results shown arethe absolute change in a component, and hencedo not discriminate between positive and negativechanges in a channel.

2.1.1. Red channelThe change in the red channel was analysed for

the dot arrays (Fig. 5, 2a). It can be clearly seenfrom the chart that the greatest change in the redcomponent was observed in the centre block ofdots, which correspond to the red dots in Fig. 2a.

2.1.2. Green channelBy using the same software, the arrays were ana-

lysed for change in green component (Fig. 5, 2b).The chart shows that for all of the dots that had beenchanged, there was a large change in the greencomponent. For the dots that remained unchanged( those at the edge of the array), there was nochange.

2.1.3. Blue channelSimilarly, the arrays were analysed for changes in

the blue channel (Fig. 5, 2c). It is seen clearly thatthe greatest change is observed in the red dots,since the blocks of darker blue dots would bemore similar to the original aqua colour.

Fig. 4. Flowchart showing steps in data processing from data in to data out.

Fig. 3. 7U7 dot array produced by Kodak.


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2.1.4. Multi-channel responseTo observe the `overall' colour change, the

change in trichromatic values was plotted (Fig. 5,3).

2.2. 96-Well microplate

96-Well microplates are widely used in analyticaland bioanalytical measurements. In bioanalyticalapplications, there is a trend towards higher andhigher density microplates. For example, GreinerLabortechnik has produced microplates with 384and 1536 wells each [ 12 ]. Existing technology forreading colour intensity of plate wells involves pri-marily the moving of a light source and detectoracross the wells. In the case of high density wells,this proves an obstacle to increasing the number ofwells, as locating each well proves more dif¢cult.By adopting the digital image approach, this prob-lem may be tackled since the locating of each well isa software function, and it helps to avoid the limi-

tations provided by the conventional plate-wellreader approach. Also, analysis times may be low-ered by taking a single image, rather than having toscan a light source and detector over a large numberof wells. Each well of the microplate is mapped byalmost 3000 pixels (and thus almost 3000 repli-cate measurements are possible) per well [ 6 ]. Anenzyme-linked immunosorbent assay (ELISA) wasperformed in the microplate with ovalbumin (OVA)as the antigen, anti-OVA as the primary antibodyand horseradish peroxidase as the substrate, pro-ducing a red^yellow colour. The concentration ofthe OVA was 10^0.3125 mg / ml. The concentrationof the anti-OVA was 1.62^0.00316 Wl / ml. This pro-duced a checkerboard with a changing colourintensity in two dimensions. This was then analysedby the same setup as previously mentioned. Theresults are shown in Fig. 6. Once again, these resultsdemonstrate the ability of the digital camcorder toprocess large amounts of data quickly and to detectsmall changes in colour.

Fig. 5. Analysis of changes in red, green, blue and trichromatic values.


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3. Conclusions

The application of digital imaging as a detectorfor colourimetric reactions has great potential formany applications which involve the productionof chemical colour changes. The high spatial reso-lution of the digital images which can be producedby modern digital cameras facilitates the use ofextremely small reagent areas, which can save onexpensive reagents. The possibilities for analysingmany hundreds of dots, each being its own singlereaction, are greatly aided by this system. Thisapproach has already been adopted in drug discov-ery, and Perkin Elmer Life Sciences have a CCDimaging system based on low level luminescenceand £uorescence which is useful for imaging indrug discovery [ 13 ].

The implications to generic analytical measure-ments include tiny sample volumes (Wl or sub-Wlrange), multiple replicate measurements per dot /well, assay replication and multicomponent analy-sis. Many modern digital cameras can provide ahigh spatial resolution in the order of approxi-mately 1.5 M pixels per image, for example theKodak DC265 Digital Camera can provide 1.6megapixel resolution [ 14 ]. Each pixel can be con-sidered a sensor which provides its own set of dataabout the sample being analysed. Considering animage of a surface which is 50% active, one couldhave approximately 750 000 analytical measure-ments performed. If analysis of 100 componentswas required, one could still achieve 7500 replicatemeasurements. At present, an 8-bit resolution isbeing used to analyse the colour of the dots. Higher

bit resolution (12-bit or 16-bit colour measure-ment ) could provide better analytical resolutionand allow for increased sensitivity to colourchanges. However, an increased quantity of datahas the drawback of producing an increased ¢lesize to be stored. In systems where images areacquired and analysed rapidly, this provides anobvious limitation in terms of the storage spacerequired for the data set. Data mining to extractthe necessary information from the data is impera-tive if such a high resolution system is to provepractical.

Further possibilities can be seen by the use ofmonochrome light to illuminate the microdotarray coupled with the use of a simple black andwhite camera. Bene¢ts of this are improved sensi-tivity, achieved by aligning the wavelength of theilluminating light with the dye's absorption peak, aswell as reduced cost of instrumentation.

National Instruments LabVIEW0 and IMAQVision0 have been shown to be useful tools fordeveloping software for pattern recognition andprocessing. The £exibility of these packages willallow for applications to be customised and agreater level of automation included.

Acknowledgements

The authors wish to gratefully acknowledge thesupport and hard work of the research teams ofboth the Kodak UK Process Measurement groupand the National Centre for Sensor Research at Dub-lin City University. Thanks are also due to Dr ShaneO'Neill for his assistance with the LabVIEW0 pro-gramming. Financial support from the Higher Edu-cation Authority and Enterprise Ireland is gratefullyacknowledged.

References

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[ 4 ] P.M. Epperson, J.V. Sweedler, R.B. Bilhorn, G.R. Sims,M.B. Denton, Anal. Chem. 60 (1988) 327A.

[ 5 ] S. Hislip, G. Pennarun Thomas, B. Manning, R. O'Ken-nedy, S. Edwards and D. Diamond, Proceedings ofSPIE, 3856, 1999.

Fig. 6. Trichromatic change for ELISA OVA checkerboard.


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[ 6 ] E.J. Giorgianni and T.E. Madden, Digital Color Manage-ment: Encoding Solutions, Addison Wesley, USA, 1998,p.9.

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[ 10 ] E. Brynda, M. Houska, J. Síkvor, J.J. Ramsden, Biosens.Bioelectron. 13 (1998) 165.

[ 11 ] National Instruments (online), available from: http: / /www.ni.com (accessed 31 January 2000).

[ 12 ] Greiner Labortechnik (online), available from http: / /www.greiner-lab.com (accessed 3 February 2000).

[ 13 ] Perkin Elmer Life Sciences (online), available fromhttp: / /www.wallac.com (accessed 3 February 2000).

[ 14 ] Kodak DC265 homepage (online), available from:http: / /www.kodak.com /US /en /digital /cameras /dc265 (accessed 3 February 2000).

Liam Byrne and John Barker are postgraduate students and GaeëllePennarun-Thomas is a post-doctoral research fellow at the School ofChemical Sciences at Dublin City University. Their research, carriedout at the National Centre for Sensor Research, encompassesaspects such as digital image processing, software programmingand development of novel methods for analytical measurementand processing.

Steve Edwards is head of the Engineering Technologies group ofKodak Research Division Europe, at Harrow in Middlesex. He isparticularly interested in rapid analytical measurements and sensorapplications, both electrochemical and optical.

Professor Dermot Diamond is a senior lecturer at the School ofChemical Sciences at Dublin City University. He is currently theResearch Director of the DCU branch of the Biomedical andEnvironmental Sensor Technology (BEST) Centre. His researchinterests include development of novel ISE systems, micro-instrumentation, sensor arrays and virtual instruments.


Digital imaging as a detector for generic analytical measurements

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